Subunit Communication within Dimeric SF1 DNA Helicases

Binh Nguyen; John Hsieh; Christopher J Fischer; Timothy M Lohman

doi:10.1016/j.jmb.2024.168578

Subunit Communication within Dimeric SF1 DNA Helicases

J Mol Biol. 2024 Apr 20;436(11):168578. doi: 10.1016/j.jmb.2024.168578. Online ahead of print.

Authors

Binh Nguyen¹, John Hsieh², Christopher J Fischer³, Timothy M Lohman⁴

Affiliations

¹ Department of Biochemistry & Molecular Biophysics, Washington University School of Medicine, 660 S. Euclid Ave, Saint Louis, MO 63110, USA.
² Department of Biochemistry & Molecular Biophysics, Washington University School of Medicine, 660 S. Euclid Ave, Saint Louis, MO 63110, USA; Biochemistry & Biophysics, Blueprint Medicines, Cambridge, MA 02139, USA.
³ Department of Physics and Astronomy, University of Kansas, Lawrence, KS 66045, USA.
⁴ Department of Biochemistry & Molecular Biophysics, Washington University School of Medicine, 660 S. Euclid Ave, Saint Louis, MO 63110, USA. Electronic address: lohman@wustl.edu.

PMID: 38648969
DOI: 10.1016/j.jmb.2024.168578

Abstract

Monomers of the Superfamily (SF) 1 helicases, E. coli Rep and UvrD, can translocate directionally along single stranded (ss) DNA, but must be activated to function as helicases. In the absence of accessory factors, helicase activity requires Rep and UvrD homo-dimerization. The ssDNA binding sites of SF1 helicases contain a conserved aromatic amino acid (Trp250 in Rep and Trp256 in UvrD) that stacks with the DNA bases. Here we show that mutation of this Trp to Ala eliminates helicase activity in both Rep and UvrD. Rep(W250A) and UvrD(W256A) can still dimerize, bind DNA, and monomers still retain ATP-dependent ssDNA translocase activity, although with ∼10-fold lower rates and lower processivities than wild type monomers. Although neither wtRep monomers nor Rep(W250A) monomers possess helicase activity by themselves, using both ensemble and single molecule methods, we show that helicase activity is achieved upon formation of a Rep(W250A)/wtRep hetero-dimer. An ATPase deficient Rep monomer is unable to activate a wtRep monomer indicating that ATPase activity is needed in both subunits of the Rep hetero-dimer. We find the same results with E. coli UvrD and its equivalent mutant (UvrD(W256A)). Importantly, Rep(W250A) is unable to activate a wtUvrD monomer and UvrD(W256A) is unable to activate a wtRep monomer indicating that specific dimer interactions are required for helicase activity. We also demonstrate subunit communication within the dimer by virtue of Trp fluorescence signals that only are present within the Rep dimer, but not the monomers. These results bear on proposed subunit switching mechanisms for dimeric helicase activity.

Keywords: Helicase activation; Rep; UvrD; gain of function; single molecule fluorescence.